Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of signal processing of a variety of central and peripheral neurons. For example, CaCC contributes to signal amplification of sensory inputs and regulation of excitability of both sensory and central neurons. The long- term objectives are to understand how these channels work, and how they regulate neuronal activity. Reflecting an intense interest in CaCCs as potential therapeutic targets for hypertension, cystic fibrosis and other diseases, there have been extensive efforts to determine the molecular identity of CaCCs. Because the channel properties and expression patterns of several reported molecular candidates do not match those for native CaCCs, several years ago we began the undertaking for expression cloning, leading to the identification of Xenopus and mouse TMEM16A, as well as mouse TMEM16B as CaCC subunits. In 2008, two concurrent studies were published around the same time as ours, and all three reached the same conclusion that mammalian TMEM16A corresponds to CaCC. By now, several studies of TMEM16A knockout mice have shown that TMEM16A is required for CaCC in exocrine glands and airway epithelia. With the TMEM16 family of "transmembrane proteins with unknown function" emerging as a novel family of ion channels, even the most basic questions are open and now amenable to molecular and genetic studies: How does calcium activate CaCC? How many TMEM16A subunits are present in a CaCC channel? Does TMEM16A correspond to the CaCC in sensory neurons of the dorsal root ganglion (DRG)? Is TMEM16A up regulated following denervation and, if so, does it influence nerve regeneration and/or neuropathic pain? Denervation causes up regulation of CaCC of DRG neurons - one of the best examples of neuronal CaCC, hence one specific aim of this proposal is to examine the involvement of TMEM16A in CaCC of DRG neurons with or without sciatic nerve lesion, and to explore potential roles of TMEM16A in pain sensitivity and neuropathic pain, which develops after nerve injury or in diseases like diabetes, herpes, and cancer. To better understand how CaCC channel traffic and activity may be controlled by cytosolic calcium, we will carry out biochemical and mutagenesis studies of TMEM16A, which can be heterogeneously expressed to generate CaCC. We will also use a combination of approaches to determine the CaCC stoichiometry - an important question for better appreciation of CaCC function and regulation, and the diversity of CaCCs. PUBLIC HEALTH RELEVANCE: Calcium-activated chloride channels (CaCCs) serve important physiological functions including modulation of signal processing of neurons in the central and peripheral nervous system. Having recently established the TMEM16 family of "transmembrane proteins of unknown function" as a novel ion channel family that includes TMEM16A and TMEM16B as CaCC subunits, we propose to use heterologous expression systems to study how CaCC channels work. We will also characterize CaCC endogenous to the dorsal root ganglion (DRG) and examine the role of TMEM16A in pain sensitivity and neuropathic pain. Bearing in mind that CaCC of DRG sensory neurons is up regulated after denervation, we have designed our experiments to lay the groundwork for future studies of the potential roles of CaCC in nerve regeneration and/or neuropathic pain, which develops after nerve injury, in diseases like diabetes, herpes zoster injection and cancer, and may also be induced by chemotherapy.